Use of the long pentraxin PTX3 for the treatment of diseases caused by an altered activation of the growth factor FGF-2

ABSTRACT

The use of the long pentraxin PTX3 (PTX3) or one of its functional derivatives is described for the preparation of medicament which inhibits the biological activity of the growth factor FGF-2, useful for the prevention and treatment of diseases brought about by an altered activation of said growth factor FGF-2.

This application is the U.S. national phase of international applicationPCT/IT01/00563 filed 8 Nov. 2001, which designated the US.

The present invention relates to the use of the long pentraxin PTX3(PTX3) or one of its functional derivatives for the preparation of amedicament which inhibits the biological activity of the growth factorFGF-2, said medicament being useful for the prevention and treatment ofdiseases brought about by an altered activation of said growth factorFGF-2.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 show that FGF-2 (18.000 D) is eluted from the column with aretention volume of approximately 22 ml.

FIG. 2 demonstrates that FGF-2 immobilised on plastic wells is able tobind biotinylated PTX3 in a way similar to the physiologic PTX3 ligandClq.

FIG. 3 shows that FGF-2 binds plastic immobilised PTX3 and that thisbinding is specific since FGF-2 reacts poorly with CRP and does notreact with other immobilised proteins.

FIG. 4 demonstrates that cold FGF-2 and soluble PTX3 inhibited theinteraction between ¹²⁵I-FGF-2 and plastic immobilised PTX3.

FIG. 5 demonstrates that FGF-2 binds PTX3 in a saturable way and withelevated affinity (Kd =8-16 nM).

FIG. 6 shows that PTX3 is able to inhibit in a dose-dependent manner thebinding of FGF-2 to its high-and low-affinity receptors on endothelialcells.

FIG. 7 shows that PTX3 inhibits the mitogenic activity exerted onendothelial cells by FGF-2 in a dose-dependent manner with an ID₅₀ equalto 30 nM.

FIG. 8 shows that PTX3 is able to block the autocrine loop ofstimulation induced by FGF-2 in MAE3F2T cells, inhibiting their FGF-2dependent proliferation, in a manner similar to that exerted by antiFGF-2 antibodies and suramin.

FIG. 9 shows that PTX3 is able to block the neovascularization inducedin vivo by FGF-2.

FIG. 10 shows that PTX3 over-expression inhibits FGF2MAE3F2T cellsproliferation and that the extent of this inhibition correlates with theamount of PTX3produced by the different clones tested.

FIG. 11 shows that the invasive capacity of PTX3 over-expressingFGF2MAE3F2T clones is significantly lower that that of parentalFGF2MAE3F2T cells.

Among the diseases brought about by an altered activation of the growthfactor FGF-2 are included diseases provoked by an altered angiogenesisand by an uncontrolled proliferation of fibroblasts or smooth musclecells.

The first compound endowed with antiangiogenic activity was discoveredin the cartilage by Henry Brem and Judath Folkman in 1975 (J. Exp. Med.Feb. 1, 1975;141(2):427-39). The authors thought that this discoverycould be used to control pathological process, such as tumor growth,metastasis, chronic and acute inflammation of the joint and diabeticretinopathy, using selective inhibitors of the pathologicalneoangiogenesis.

Angiogenesis in the adult is normally quiescent, but it represents anormal function, for example in the healing of wounds, or in thereconstruction of the endometrium during the female reproductive cycle.

The angiogenic response is physiologically stimulated when the vascularfunctions are reduced and tissue perfusion is inadequate.

More generally, it can be claimed that, in physiological conditions,angiogenesis constitutes a positive feedback in response to inadequateperfusion, or to a reduced supply of oxygen and nutrients, such asoccurs, for instance, in the case of occlusion of an artery, insituations of tissue mass growth (for example, the neovascularisationthat accompanies the formation of muscle tissue); and in the case of anincreased work load in association with an increased oxygen and nutrientrequirement.

It is well known that the growth of a primary tumor is favoured by goodvascularisation of the tumor tissue. An adequate supply of oxygen andnutrients promotes rapid growth of the tumor itself.

It has been demonstrated that the extent of angiogenesis can be anextremely negative factor in the prognosis of neoplasms (van Hinsbergh VW, Collen A, Koolwijk P; Ann. Oncol., 10 Suppl., 4:60-3, 1999;Buolamwini J K; Curr., Opin., Chem., Biol., 3(4):500-9, August 1999).

It is also known, in the tumor field, that a fundamental stage in thebiology of the tumor cell is the acquisition of metastasisingcapability.

The tumor cells that metastasise are able to lose adherence to thesurrounding structures, invade blood and lymphatic vessels and coloniseother tissues at a distance where they can continue to reproducethemselves.

Metastasising is also a critical event in the clinical history of thedisease, being the main cause of death due to cancer. It is closedassociated with and facilitated-by the presence of vascular tissue inthe tumor site or adjacent areas.

The migration of tumor cells across the surrounding structures enablesthe cells to reach the intra-tumor blood vessels, whether pre-existingor formed by neo-angiogenesis, and thus reach the bloodstream (Ray J M.,Stetler-Stevenson W G; Eur. Respir. J., 7(11):2062-72, 1994;Stetler-Stevenson W G, Liotta L A, Kleiner D E Jr; FASEB J.,7(15):1434-41, December 1993).

The presence of communication between lymphatic and blood vessels in thevascular region of the tumor enables the neoplastic cells to move inboth vascular systems.

Recent studies have shown a direct relationship between angiogenesis andarthritic disease (Koch A E; Arthritis and Rheumatism 41:951-962, 1998).In particular, it has been demonstrated that neo-vascularisation of thearticular cartilages plays a crucial role in pannus formation and inprogression of arthritis. A normal cartilage does not possess bloodvessels, while the synovial fluid of arthritic patients contains anangiogenesis-stimulating factor produced by endothelial cells (EASF).

The presence of this factor is associated with vascularisation anddegradation of the cartilage.

Other diseases are also related to abnormal angiogenesis.

It has been found that, in diabetic retinopathy [Histol Histopathol.October 1999; 14(4): 1287-94], psoriasis [Br J Dermatol. December 1999;141(6):1054-60], chronic inflammation and arteriosclerosis [Planta MedDecember 1998; 64(8):686-95], neovascularisation of the affected tissuesis a facilitating factor.

The growth factor FGF-2 is a cationic polypeptides of 18 kDa capableboth of inducing neoangiogenesis in vivo and cells proliferation,chemotaxis and production of proteases in endothelial cells in culture(Basilico and Moscatelli, 1992, Adv. Cancer Res. 59:115-65).

The role of FGF-2 in the tumoral angiogenesis is already known (Pathol.Biol. (Paris) April 1999; 47(4):364-7).

In J. Pathol. September 1999; 189(1):72-8 is reported that an increasedexpression of FGF-2, in patients suffering from mesotelioma, is relatedto an higher aggressiveness and higher mortality of patients affected bysuch tumoral disease.

In Oncogene Nov. 18, 1999;18(48):6719-24 is reported that FGF-2 confersmetastasising propriety to rat bladder carcinoma cells.

Int. J. Cancer May 5, 1999;81(3):451-8 reports that FGF-2 has a strongmitogenic activity, and is involved in the development of human tumorsand in the increases of their malignity.

For the cure of tumoral diseases the antiangiogenic therapy respect tothe standard traditional chemotherapy presents the following advantages(Cancer Research 1998, 58, 1408-16):

-   1. Higher specificity: it has as target the tumor neovascularisation    process;-   2. Higher biodisponibility: it has as target the endothelial cells,    easy reachable without the problems linked to the traditional    chemotherapeutic approach which act directly on the tumoral cells;-   3. Minor chemoresistence: this may be the most important advantage    of this therapy; in fact, being the endothelial cells, respect to    the tumoral cells, genetically stable, phenomenon of drug resistance    are improbable;-   4. Minor metastasising: the blockage of the neovascularisation    limits the propagation of the tumor cells in the other parts of the    body through the bloodstream;-   5. The apoptosis is favoured: the blockage of the vascular net in    the tumor decrease the oxygen and nutrient availability for the    tumoral cells, in this way the apoptosis is increased;-   6. Reduction of the systemic toxicity: toxic effect, such as    mielosuppression, gastrointestinal effects and transient alopecia,    present with the traditional chemotherapy, are not observed with the    antiangiogenic therapy.

To determinate these effects FGF-2 reacts with two different receptorclasses present on target cells surfaces: the receptors having highaffinity endowed with tirosin kinases activity (FGFRs) and receptorsendowed with low affinity represented by proteoglycan eparan sulfate(HSOGs) (Johnson and Williams, 1993, Adv. Cancer Res. 60:1-41;Moscatelli, 1987, J. Cell. Physiol. 131:123-30).

FGF-2 is a potent mitogen for medial smooth muscle cells and isnecessary for their proliferation after balloon catheter injury (J.Biol. Chem. Apr. 14, 2000; 275 (15): 11270-7).

Therefore, diseases caused by an altered activation of the growth factorFGF-2 also include the hyperplasia of the muscular wall of the arteriesthat occurs during restenosis after angioplastic or “coronary stenting”[J. Vasc. Surg. February 1997; 25(2):320-5].

The control of FGF-2 dependent neovascularisation represents one of thefundamental elements for the control and cure of diseases linked to analtered angiogenesis, as well as the control of FGF-2-dependentuncontrolled proliferation of fibroblasts or SMCs is crucial for thetreatment of cicatrization linked to excessive fibroblastic response andrestenosis after angioplastic.

The availability of new therapeutical compound which specificallyinhibit the biological activity of FGF-2 is an objective of primaryimportance for the prevention and treatment of diseases brought about byan altered activation of this growth factor. Such diseases includearthritic disease, tumor, tumor metastasis, diabetic retinopathy,psoriasis, chronic inflammation, arteriosclerosis, cicatrization linkedto excessive fibroblastic response and restenosis after angioplastic.

PTX3 is expressed in various cell types (Bottazzi et Al., J., Biol.Chem. 1997, 272: 32817-32823) particularly in mononuclear phagocytes andendothelial cells, after exposure to the inflammatory cytokinesInterleukin 1β (IL-1β) and Tumor Necrosis Factor alpha (TNF-α).

To date, the biological function of PTX3 has not yet been fullyunderstood.

PTX3 consists of two structural domains, a N-terminal domain unrelatedto any known molecule, and a C-terminal domain similar to the shortpentraxins such as C-reactive protein (CRP) (Breviario F., et al., J.Biol. Chem. 267:22190, 1992).

A substantial degree of similarity has been found between human PTX3(hPTX3) and animal PTX3s. In particular, mouse PTX3. (mPTX3) is verysimilar to hPTX3 in terms of DNA sequence and gene organisation andlocation. The degree of identity between human and murine PTX3 gene is82%, and reaches 90% if conservative substitutions are considered(Introna M., et al.: Blood 87 (1996) 1862-1872). The murine PTX3 gene islocated on chromosome 3 of the mouse in a region similar to the human 3qregion (q24-28), in agreement with the documented location of hPTX3 inthe 3q 25 region (Introna M., et al.: Blood 87 (1996) 1862-1872).

The high degree of similarity between hPTX3 and mPTX3 sequences is asign of the high degree of conservation of pentraxins during evolution(Pepys M. B., Baltz M. L.: Adv. Immunol. 34:141, 1983).

For a review about pentraxins see H. Gewurz et al, Current Opinion inImmunology, 1995, 7:54-64.

The international application WO99/32516, filed in the name of theapplicant, describes the use of the long pentraxin PTX3 for thetreatment of infective diseases, inflammatory or tumoral. The anti-tumoractivity shown by PTX3, described in WO99/32516, is mediated by theleucocitary recruitment, i.e. on immunologic bases. In WO99/32516 it isnever described or suggested the use of PTX3 as useful agent for thetreatment of diseases associated with an altered activation of thegrowth factor FGF-2.

U.S. Pat. No. 5,767,252 describes a neuronal growth factor belonging tothe pentraxin family (see also the references there cited). This patentrefers to the neurobiology sector.

It has now been found that the long pentraxin PTX3 is capable to bindthe FGF-2 with high affinity and specificity. The binding of PTX3 withFGF-2 (K_(d)=8-16 nM) prevents the binding of the latter to its highaffinity tyrosine-kinase receptors, and the binding at the site of lowaffinity receptors, represented by the eparan-sulphate proteoglycans,present on the cell surface. The inhibition of the binding causes aninhibition of the FGF-2 biological activity.

The interaction between FGF-2/PTX3 depends on a correct conformationalstructure of the growth factor, not only on its basic nature, since PTX3does not bind the cytocrome C (a protein that share with the FGF-2molecular weight (18 kDa) and isoelectric point (pH 9.6)).

Moreover, the C-reactive protein, homologous to PTX3, does not bindFGF-2.

Is therefore object of the present invention the use of the longpentraxin PTX3 or a derivatives thereof, or its domain, for preparing amedicament for the prevention and cure of diseases which arecounteracted by the inhibition of the biological activity of the growthfactor FGF-2.

A further object of the present invention is the use of the longpentraxin PTX3 or a derivatives of the PTX3 or its domain, for preparinga medicament for the prevention and cure of diseases brought about by analtered activation of the growth factor FGF-2. A further object of thepresent invention is the use of the long pentraxin PTX3 or a derivativethereof, or its domain, for preparing a medicament for the prevention orcure of diseases brought about by an altered angiogenesis, in which thealtered angiogenesis is provoked by an altered activation of the growthfactor FGF-2, example of said diseases are: arthritic disease, tumormetastasis, diabetic retinopathy, psoriasis, chronic inflammation,arteriosclerosis or tumor, in which the tumor is, for example, sarcoma,carcinoma, carcinoid, bone tumor or neuroendocrine tumor.

A further object of the present invention is the use of the longpentraxin PTX3 or a derivatives of the PTX3 or its domain, for preparinga medicament for the treatment of diseases associated with uncontrolledFGF-2-dependent proliferation of fibroblasts or smooth muscular cells,such as the cicatrization linked to excessive fibroblastic response, andthe restenosis after angioplastic.

The compound according to the invention is suitable to be used for theinhibition of FGF-2 activity in target cells not only when it isadministered as recombinant protein, but also when it is administeredendogenously in consequence of the gene transfer of its cDNA.

Is therefore a further object of the present invention the use of cDNAfull length of human PTX3 or its derivative or its domain, for preparingplasmidic or viral expression vectors comprising said cDNA, for the genetherapy of diseases caused by an altered activation of the growth factorFGF-2, in which the diseases are, for example, tumor, tumor metastasis,cicatrization linked to excessive fibroblastic response or restenosisafter angioplastic.

For “long pentraxin PTX3” is intended any long pentraxin PTX3,independently from its origin natural (human or animal) or synthetic.

For derivatives is intended any functional analogous of the longpentraxin PTX3 as above defined that bear at least a mutation,maintaining its functional capacity to selectively inhibit the FGF-2.

The preferred long pentraxin PTX3 is the human PTX3, which sequence isdescribed in WO99/32516.

The long pentraxin PTX3 described herein can also be used in combinationwith one or more anticancer drugs for the treatment of tumor diseases inwhich the increased expression of FGF-2 provokes an higheraggressiveness of the tumor disease, or an higher metastasisingcapability.

In fact, it is well known that, to avoid the unwanted side effectsmaintaining the same therapeutic efficacy, most oncological patients aretreated not with a single anticancer drug, but with a combination ofseveral anticancer agents or with a combination of anticancer agents inassociation with antiangiogenic compounds.

The mechanism of action of the more usual anticancer drugs is completelydifferent from the mechanism of action of the compound according to thepresent invention, in fact, the usual anticancer drugs are cytotoxic vstumoral cells.

The compound according to the present invention having a differentmechanism of action, exert a curative effect (adjuvant effect)additional to the effect exerted by the anti-tumor drug to which it isassociated.

It is therefore a further object of the invention described herein thecombination of the long pentraxin PTX3 with one or more known anticancerdrugs.

A further object of the invention described herein is a pharmaceuticalcomposition containing the long pentraxin PTX3 in combination with oneor more known anticancer drugs, such as alkylating agents, topoisomeraseinhibitors, antitubulin agents, intercalating compounds,anti-metabolites, natural products such as vinca alkaloids,epipodophyllotoxins, antibiotics, enzymes, taxans, andcyto-differentiating compounds, and one or more excipients or vehiclespharmacologically acceptable.

A further object of the invention described herein is the use of thelong pentraxin PTX3, in combination with one or more known anti-tumorcompounds, for preparing a medicament for the treatment of tumor, inwhich the increased expression of FGF-2 provokes an higheraggressiveness of the tumor.

A further object of the invention described herein is the use of thelong pentraxin PTX3, in combination with one or more known anti-tumorcompounds, for preparing a medicament for the prevention of the onset oftumor metastasis, in which the increased expression of FGF-2 provokes anhigher metastasising capability.

A further object of the invention described herein is the use of thelong pentraxin PTX3 in combination with, the anticancer compound, forpreparing a medicament for the treatment of tumor, characterised in thatthe long pentraxin PTX3 is present as adjuvant of the anticancercompound.

The following examples illustrate the invention.

EXAMPLE 1 Capacity of PTX3 to Bind FGF in Solution.

This experiment has been performed in order to evaluate PTX3 binding toFGF-2. PTX3 has been produced as described by Bottazzi et al., 1997, J.Biol. Chem. 272:32817-32823. Human recombinant FGF-2 has been producedas described by Isacchi A. et al., Proc. Natl. Acad. Sci. U.S.A. (1991),88:2628-32. When necessary, FGF-2 has been labelled with ¹²⁵I followingthe method described by Isacchi et al. (see below).

Experimental Procedure

100 μl of PBS containing 1 μg of FGF-2, (or alternatively 1 μg of PTX3)were chromatographed on a size exclusion fast protein liquidchromatography Superose 12 column (Pharmacia): this column is able toseparate proteins depending on their molecular weight. The column waseluted with PBS at a flow rate equal to 1 ml/minute and 1 ml fractionswere collected and analyzed by dot, blot with specific antibodies forthe two proteins. In order to evaluate PTX3 binding to FGF-2 the twoproteins (5 g and 1 μg, respectively), were mixed in 100 μl PBS andincubated at 4° C. for 10 min. before loading onto the column. Fractionswere collected and analysed by dot-blot with anti FGF-2 specificantibodies. The results reported in FIG. 1 show that FGF-2 (18.000 D) iseluted from the column with a retention volume of approximately 22 ml.In the same conditions PTX3 (450.000 D) is eluted with the void volumeof the column (7 ml.) When FGF-2 pre-incubated with PTX3 for. 10 min. at4° C. was loaded onto the column, a change in its chromatographicbehaviour was observed: in this experimental conditions FGF-2 was elutedfrom the column with a retention volume equal to PTX3 alone.

EXAMPLE 2 Capacity of Biotinylated PTX3 to Bind Plastic ImmobilisedFGF-2

100 μl of NaHCO₃ pH 9.6 containing 500 ng of FGF-2 or of the complementcomponent C1q were incubated 18 h at 4° C. in 96 wells plastic plates.At the end of incubation wells were washed 3 times with PBS andsubsequently incubated 2 h at room temperature with 200 μl of PBScontaining 1 mg/ml of Bovine Serum Albumin (BSA). At the end ofincubation wells were washed again 3 times with PBS and incubated with30 ng/ml of ¹²⁵I-FGF-2 in presence of increasing amounts of biotinylatedPTX3 in 100 μl of PBS (2 h at room temperature). Alternatively wellswere adsorbed with increasing doses of FGF-2 or C1q and incubated with100 ng/ml of biotinylated PTX3 in 100 μl of PBS. After this incubationwells were washed 3 times with PBS and incubated 1 h at room temperaturewith 100 μl of streptavidin HRP conjugate ( 1/2000). Reaction wasdeveloped by adding the chromogen microwell peroxidase substrate systemABTS (Kirkegaard & Perry Laboratories). Plates were read in an automaticELISA reader at 405 nm. Results reported in FIG. 2 demonstrate thatFGF-2 immobilised on plastic wells is able to bind biotinylated PTX3 ina way similar to the physiologic PTX3 ligand C1q (Bottazzi B. et al.,1997, J. Biol. Chem. 272:32817-32823).

EXAMPLE 3 Characterisation of PTX3-FGF-2 Interactions

96 wells plates were coated at 4° C. with 100 μl of NaHCO₃ pH 9.6containing PTX3 or C reactive protein (200 nM) or alternatively 2 μg/mlof the following proteins used as negative control: bovine serumalbumin, fibronectin, gelatine or laminin. After incubation, wells werewashed 3 times with PBS and blocked 2 h at room temperature with 200 μlof 1 mg/ml of BSA in PBS. At the end of incubation wells were washed 3times with PBS and subsequently incubated with 30 ng of ¹²⁵I-FGF-2 for 2h at room temperature. Wells were subsequently washed 3 times with PBSand bound ¹²⁵I-FGF-2 was recovered by washing each well twice with 200μl of 2% SDS in water. Levels of ¹²⁵I-FGF-2 were measured in a gammacounter. Results reported in FIG. 3 shows that FGF-2 binds plasticimmobilised PTX3 and that this binding is specific since FGF-2 reactspoorly with CRP and does not react with other immobilised proteins.

EXAMPLE 4 ¹²⁵I-FGF-2 Binding to Plastic Immobilised PTX3 in the Presenceof an Excess of Cold FGF-2

In a second set of experiments the binding of ¹²⁵I-FGF-2 to plasticimmobilised PTX3 has been tested in the presence of an excess of bothnative or heat denatured cold FGF-2 (100 nM), in the presence of similarCytocrome C concentrations or in the presence of an excess of solublePTX3 (300 nM). The experiment was performed as described above: Theresults reported in FIG. 4 demonstrate that cold FGF-2 and soluble PTX3inhibited the interaction between ¹²⁵I-FGF-2 and plastic immobilisedPTX3. The observation that heat-inactivated FGF-2 and Cytocrome C(having the same molecular weight and isoelectric point as FGF-2) arenot able to bind PTX3 suggests that a correct conformation of the growthfactor rather than its basic nature is involved in its capacity to bindPTX3.

EXAMPLE 5 Dissociation Constant (Kd) of FGF-2/PTX3 Interaction

With this experiment the dissociation constant (Kd) FGF-2/PTX3interaction has been determined. For this purpose increasing doses of¹²⁵I-FGF-2 were incubated with plastic immobilised PTX3 and bindingresults were analysed by Scatchard plot. Results reported in FIG. 5demonstrate that FGF-2 binds PTX3 in a saturable way and with elevatedaffinity (Kd=8-16 nM). This affinity is similar to the affinitypreviously calculated for the interaction of PTX3 with its physiologicalligand C1q.

EXAMPLE 6 Effect of PTX3 on FGF-2 Binding to Endothelial Cells

Transformed foetal bovine aortic endothelial cells GM7373 were seeded at80,000 cells/cm² in 24-well dishes in Eagle's minimal essential medium(Eagle's MEM) containing 10% FCS. After 24 h at 37° C. adherent cellswere washed twice with Eagle's MEM without FCS and subsequentlyincubated for 2 h at 4° C. with ¹²⁵I-FGF-2 (10 ng/ml) in Eagle's MEMcontaining 0.15% gelatine and 20 mM HEPES pH 7.5 in the absence or inthe presence of increasing concentrations of PTX3. At the end ofincubation the amount of ¹²⁵I-FGF-2 bound to low (HSPGs) and highaffinity receptors (FGFR) was evaluated as described by Moscatelli, 1987J. Cell. Physiol. 131:123-30. Briefly cells were rinsed twice with 2 MNaCl in 20 mM HEPES buffer (pH 7.5) to remove ¹²⁵I-FGF-2 bound tolow-affinity binding sites and twice with 2 M NaCl in 20 mM sodiumacetate (pH 4.0) to remove ¹²⁵I-FGF-2 bound to high-affinity bindingsites. Non-specific binding was measured in the presence of a 100-foldmolar excess of unlabeled FGF-2 and subtracted from all the values.Results reported in FIG. 6 shown that PTX3 is able to inhibit in adose-dependent manner the binding of FGF-2 to its high- and low-affinityreceptors on endothelial cells.

EXAMPLE 7 Effect of PTX3 on Mitogenic Activity Exerted by FGF-2 onEndothelial Cells

GM 7373 cells were seeded at 75,000 cells/cm² in 48-well plates in EagleMEM containing 10% FCS. After 24 h at 37° C. adherent cells were washedtwice with Eagle's MEM without FCS and subsequently incubated for 24 hat 37° C. in Eagle's MEM containing 0.4% FCS in the absence or in thepresence of FGF-2 (10 ng/ml) and increasing concentrations of PTX3. Atthe end of incubation cells were trypsinized and counted. In a differentset of experiments GM 7373 cells were treated as described above in thepresence of different mitogenic stimuli: 10% FCS; 5 μg/mldyacyl-glycerol (DAG); 10 ng/ml phorbol ester (TPA) 30 ng/ml epidermalgrowth factor (EGF) or 30 ng/ml vascular endothelial growth factor(VEGF). Results reported in FIG. 7 shown that PTX3 inhibits themitogenic activity exerted on endothelial cells by FGF-2 in adose-dependent manner with an ID₅₀ equal to 30 nM. This value is similarto the Kd calculated for the interaction FGF-2-PTX3. These resultssuggest that the inhibition of FGF-2 biological activity by PTX3 is dueto its sequestration in extracellular sites. The inhibitory activity ofPTX3 on FGF-2 is specific since it is not detectable when other mitogensare used to induce, cellular proliferation.

EXAMPLE 8 Effect of PTX3 on the Proliferation of Transformed FoetalBovine Aortic Endothelial Cells

In this experiment we have investigated the effect of PTX3 on theproliferation of the murine aortic endothelial cell line MAE3F2T stablytransfected with an expression vector coding for FGF-2 (Gualandris etal. 1996, Cell Growth Diff. 7:147-60). These cells can proliferate inresponse to a autocrine loop of stimulation induced by endogenous FGF-2(Gualandris et al. 1996, Cell Growth Diff. 7:14760). MAE3F2T cells wereseeded at 10,000/cm² in 48-well plates in Dulbecco medium (DMEM) addedwith 10% FCS. After 24 h at 37° C., adherent cells were washed twicewith DMEM without FCS and incubated for 72 at 37° C. in DMEM containing0.4% FCS in the presence or not of PTX3 (70 nM), anti FGF-2 specificantibodies or suramin (50 μg/ml). Suramin is known for its capacity tosequestrate extracellular FGF-2 and inhibits its biological activities(Rusnati et al., 1996, Mol. Biol. Cell. 7:369-381). At the end ofincubation cells were trypsinized and counted in a Burker chamber.Results reported in FIG. 8 shown that PTX3 is able to block theautocrine loop of stimulation induced by FGF-2 in MAE3F2T cells,inhibiting their FGF-2 dependent proliferation, in a manner similar tothat exerted by anti FGF-2 antibodies and suramin.

EXAMPLE 9 Effect of PTX3 onto the Neovascularization Induced in vivo byFGF-2

The antiangiogenic potential of PTX3 was evaluated in vivo in the chickembryo chorioallantoic membrane (CAM) assay. Briefly, a window wasopened in the egg shell of 3 day-old fertilized chicken eggs. At day 8,gelatin sponges were implanted on the CAMs and adsorbed with 10 ul ofPBS alone or containing FGF-2 (at 500 ng/sponge) in the absence or inthe presence of PTX3 (5 ug/sponge) (5-6 embryos per group). After 4days, CAMs were observed in ovo under a Zeiss SR stereomicroscope andthe angiogenic response was scored by two investigators withoutknowledge of the samples tested and graded on an arbitrary scale of0-4+, with 0 representing no angiogenic response and 4+ representing thestrongest activity. Results reported in FIG. 9 shown that PTX3 is ableto block the neovascularization induced in vivo by FGF-2.

EXAMPLE 10 Gene Therapy with PTX3

Murine endothelial cells overexpressing FGF-2, named FGF2MAE3F2T(Gualandris et al. 1996, Cell Growth Diff. 7:147-60) were transfectedwith human PTX3 full length cDNA subcloned in the commercial expressionvector pLXSH (Clontech).

In vitro studies on the transfected cell lines obtained were, performedto study the effect of PTX3 over-expression on FGF-2-dependentproliferation of FGF2MAE3F2T cells and their invasive behaviour onthree-dimensional fibrin gels.

In details, several FGF2MAE3F2T, clones expressing different levels ofPTX3 and parental FGF2MAE3F2T cells (that do not produce PTX3) wereseeded at 10,000/cm² in 48-well plates in Dulbecco medium (DMEM) addedwith 10% FCS. After 24 h at 37° C., adherent cells were washed twicewith DMEM without FCS and incubated for different period of time at 37°C. in DMEM containing 0.4% FCS. At the end of incubations cells weretrypsinized and counted in a Burker chamber. Results reported in FIG.10, shown that PTX3 over-expression inhibits FGF2MAE3F2T cellsproliferation and that the extent of this inhibition correlates with theamount of PTX3 produced by the different clones tested.

To evaluate the invasive behaviour of FGF2MAE3F2T clones over-expressingPTX3 on three-dimensional fibrin gels, cell aggregates were prepared onagarose-coated plates exactly as described (Gualandris et al. 1996, CellGrowth Diff. 7:147-60). These aggregates were seeded onto fibrin-coated48 well-plates. Immediately after seeding, 250 l of calcium-free mediumcontaining fibrinogen (2.5 mg/ml) and thrombin (250 mU/ml) were added toeach well and allowed to gel for 5 min at 37° C. Then, 500 l of culturemedium were added on the top of the gel. In all the, experiments, thefibrinolytic inhibitor trasylol was added to the gel and to the culturemedium at 200 KIU/ml to prevent the dissolution of the substrate.Formation of radially growing cell sprouts was evaluated after 2 days bycomputerized image analysis.

Results reported in FIG. 11 shown that the invasive capacity of PTX3over-expressing FGF2MAE3F2T clones is significantly lower that that ofparental FGF2MAE3F2T cells.

The results obtained demonstrate that PTX3 inhibits FGF-2 activity inendothelial cells when it is endogenously produced after gene transferof its cDNA.

Thus, the compound according to the invention can be used in genetherapy protocols in accordance, to known methods (In Vivo.January-February 1998; 12(1):59-67; In Vivo. January-February 1998;12(1):35′-41; In Vivo. November-December 1994; 8(5):771-80) for thetreatment of diseases caused by an altered activation of the growthfactor FGF-2.

1. A method of treating a disease characterized by uncontrolledproliferation of fibroblasts or smooth muscle cells, wherein the diseaseis selected from the group consisting of cicatrization, restenosis afterangioplasty, diabetic retinopathy, psoriasis and atherosclerosis; saidmethod comprising administering full length human or mouse longpentraxin PTX3 to a person in need of said treatment.
 2. The method ofclaim 1, wherein said full length human or mouse long pentraxin PTX3 isnaturally occurring and administered to the person.
 3. The method ofclaim 1, wherein said full length human or mouse long pentraxin PTX3 isa synthetic PTX3 made by recombinant or chemical means and administeredto the person.
 4. The method of claim 1, wherein full length human longpentraxin PTX3 is administered.
 5. The method of claim 1, whereincicatrization is treated.
 6. The method of claim 1, wherein restenosisafter angioplasty is treated.
 7. The method of claim 1, wherein diabeticretinopathy is treated.
 8. The method of claim 1, wherein psoriasis istreated.
 9. The method of claim 1, wherein atherosclerosis is treated.10. A method of treating a disease characterized by uncontrolledproliferation of fibroblasts or smooth muscle cells, wherein the diseaseis selected from the group consisting of cicatrization, restenosis afterangioplasty, diabetic retinopathy, psoriasis and atherosclerosis; saidmethod comprising administering a domain of human or mouse longpentraxin PTX3 which binds FGF-2 to a person in need of said treatment.11. The method of claim 10, wherein cicatrization is treated.
 12. Themethod of claim 10, wherein restenosis after angioplasty is treated. 13.The method of claim 10, wherein diabetic retinopathy is treated.
 14. Themethod of claim 10, wherein psoriasis is treated.
 15. The method ofclaim 10, wherein atherosclerosis is treated.
 16. A method of treating adisease characterized by uncontrolled proliferation of fibroblasts orsmooth muscle cells, wherein the disease is selected from the groupconsisting of cicatrization, restenosis after angioplasty, diabeticretinopathy, psoriasis and atherosclerosis; said method comprisingadministering a fragment of human or mouse long pentraxin PTX3comprising its domain which binds FGF-2 to a person in need of saidtreatment.
 17. The method of claim 16, wherein cicatrization is treated.18. The method of claim 16, wherein restenosis after angioplasty istreated.
 19. The method of claim 16, wherein diabetic retinopathy istreated.
 20. The method of claim 16, wherein psoriasis is treated. 21.The method of claim 16, wherein atherosclerosis is treated.